Process for the enzymatic preparation of protected and unprotect

Details Process for the enzymatic preparation of protected and unprotect Process for the enzymatic preparation of protected and unprotect

1992-01-23

1994-04-19

Robinson, Douglas W.

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Enzymatic production of a protein or polypeptide

530335, 530338, C12P 2100, C07K 0114, C07K 0106

Patent

active

053044703

ABSTRACT:
Protected and unprotected di- or oligopeptides are synthesized by reacting an N-terminally protected .alpha.-amino acid alkyl ester or peptide alkyl ester of the formula X--E--R.sup.1 with an amino acid or a di- or oligopeptide or a derivative thereof of the formula H.sub.2 N--Q--R.sup.2 in aqueous solution in the presence of a hydrolase, and, removing protective groups from the reaction product separated from the reaction mixture, where E is the residue of an .alpha.-amino acid or of a di- or oligopeptide, R.sup.1 is lower alkyl and X is a group which carries a charge or is polar at the pH values used for the reaction and which increases the solubility by a factor >5 compared with compounds wherein X=H, Q is the residue of an amino acid or of a di- or oligopeptide, and R.sup.2 is an optionally esterified or amidated acid group. In a preferred embodiment, the peptide or (.alpha.-)amino acid ester concentration is greater than 50 mM, wherein in a particularly preferred embodiment, the concentration ranges from 100 to 1000 mM with approximately comparable nucleophile and electrophile concentrations. The preferred substrate/enzyme ratios are >10.sup.5 M/M or >10.sup.3 M/M when papain is employed. Phthalyl, maleyl or citraconyl radicals and their derivatives or N-betainyl compounds are envisaged for use radicals (X) in the electrophilic component.

REFERENCES:
patent: 5057415 (1991-10-01), Schuetz et al.
Schellenberger et al., (1988) 2884-2889, "Specific Water-Soluble Substrates for Chymotrypsin: Attempts for Compensating Diminished P.sub.1 -S.sub.1 Interactions".
Atassi et al., 546-553, "Reaction of Proteins with Citraconic Anhydride".
Chemical Abstracts, vol. 103, Oct., 1985: Jakubke et al., "Peptides", p. 587, Abst. #121,717.
Chemical Abstracts, vol. 114, Feb., 1991: Gololobov et al., "Increased Nucleophile Reactivity of Amino Acid Beta-Naphthylamides in Alpha-Chymotrypsin-Catalyzed Peptide Synthesis", p. 336, Abst. #58,418.
Hughes et al, "Identification of Two Related Pentapeptides From the Brain With Potent Opiate Agonist Activity", Nature, 258:577, (1975).
Kuhl et al, (1984) Phamazie, 39,H.4, 280.
Konnecke et al, (1985) Monat. Chem., 116, 111-117.
Schellenberger et al. (1986) Biochim Biophys Acta, 869, 54-60.
Gololobov et al, (1990) Biochem. B Cophys Acta., 1041, 71-78.
Fruton (1982) Adv. Enzymol. Retato Areas Mol. Biol., 53, 239-306.
Morihara (1987) TIBTECH., 5, 164-170.
Jakubke et al, (1985) Angew. Chem. Int Ed. Eng., 24(2), 85-93.
Kasche (1989) in "Proteolytic Enzymes", Begnon, Ed., IRL Press, NY, 125-143.

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